1. Field of Invention
This invention relates to crosslinked protein nanoparticles and a nanonization method for producing crosslinked protein nanoparticles.
2. Background
Protein-based applications, principally but not necessarily restricted to the use of enzymes, antibodies, receptors, hormones, and structural proteins, are well established in the biotechnology, biomedicine, pharmacy, biomaterial and cosmetics industries. Some common problems related to the application of individual proteins, such as short half-life, poor stability, poor recoverability, narrow scope of use, high cost, hydrolytic instability and poor bioavailability have been addressed, at least in part, by the use of crosslinked protein crystals and aggregates. Said crystals and aggregates, comprising of micron or greater sized particles, have provided for structurally and chemically robust, long-lived material, which have bypassed many limitations common to individual proteins and thereby ameliorated some major problems. Still, their micron and greater size has introduced an assortment of problems foreign to individual proteins, such as mass transport limitations, reduced access to catalytic centers, restricted catalytic turnover due to crosslinking, and poor bioabsorptivity. In some cases, high costs have also prevailed, despite the reusability of said preparations.
There is also a continuing drive to optimize the bioactivity, stability, shelf-life, scope of use, and bioavailability of protein products by the implementation of nanotechnology. Collectively termed herein as protein nanoparticles (i.e., protein material with a cross-sectional length under one micron in every dimension), the few examples reported to date consist of protein nanoaggregates (i.e., non-crystalline protein nanoclusters, comprising of dimers and even higher associations), and protein nanocrystals (i.e., crystalline protein nanoparticles). The preparation and utilization of novel nano-sized protein aggregates and crystals, especially of the crosslinked type, describes an area of study, touting limited success.
In view of the limited availability, scope and production constraints of protein-based products, a demand exists to realize materials, such as novel crosslinked protein nanoparticles. Analogously, a demand also exists to establish convenient preparative methodologies so that said crosslinked protein nanoparticles can be better utilized, in general, to address much of the current problems of industry and medicine. Unfortunately, the development and preparation of new crosslinked protein nanoparticles has been slowed by technical problems, for which general and facile solutions are lacking. The physico-chemical attributes notably present in nanoscale materials, such as inherently high surface energy and reactivity, is one factor that underlies these difficulties. Among the established protein-based products, only bottom-up strategies, in which individual native-state proteins are brought together, forming larger associations, have been utilized to prepare crosslinked protein nanoparticles. In contrast, no approach has reported the preparation of crosslinked protein nanoparticles by employing physical size reduction methods (i.e., top-down approaches) on larger crosslinked protein materials. Indeed, nanonization (i.e., size reduction of larger material to the nanoscale) or nanofragmentation (i.e., fragmentation of larger material to yield nanosized fragments or “nanofragments”) remains to be tested on crosslinked proteins. The commonly professed belief that proteins are easily harmed by “unnatural” process conditions is one factor that has discouraged an earlier assessment of the merit of size reduction. In addition, the act of fragmenting soft materials—implying protein-based materials—is known to become increasingly difficult as the particle size diminishes. This common perception has likely served as a second discouraging factor.